1. Field of the Invention
This invention relates to scintigraphic imaging agents and reagents for preparing said agents, methods for radiolabeling said reagents and kits containing non-radioactive reagents and other components for the convenient preparation of the scintigraphic imaging agents.
Specifically, the invention relates to scintigraphic imaging agents labeled with gamma-radiation emitting isotopes such as technetium-99m (Tc-99m), reagents that can be labeled with gamma-radiation emitting radioisotopes, methods and kits for making and radiolabeling such reagents, and methods for using said scintigraphic imaging agents to image pathologic sites in a mammalian body.
More specifically, the invention relates to scintigraphic imaging agents that are radiolabeled compounds which bind with high affinty to GPIIb/IIIa receptors on activated platelets and using such radiolabeled compounds to image sites in a mammalian body.
2. Description of the Prior Art
Thrombosis and thromboembolism, in particular deep vein thrombosis (DVT) and pulmonary embolism (PE), are common clinical conditions that are associated with significant morbidity and mortality. It has been estimated that in the U.S. approximately 5 million patients experience one or more episodes of DVT per year and that over 500,000 cases of PE occur annually, resulting in 100,000 deaths (Seabold, Society of Nuclear Medicine Annual Meeting 1990). It has also been estimated that over 90% of all pulmonary emboli arise from DVT in the lower extremities.
Anticoagulant therapy can effectively treat these conditions if applied early enough. However, such treatment is associated with risks, such as internal bleeding, that prevent their unnecessary prophylactic application. More advanced techniques of thrombolytic intervention (such as the administration of recombinant tissue plasminogen activator or streptokinase) can be used in acute cases, but these techniques carry even greater risk. Moreover, effective clinical application of these techniques requires that the site of the offending thrombus be identified so as to monitor the effect of treatment.
For these reasons, a rapid means of detecting and localizing thrombi in vivo, most preferably using non-invasive methods, is highly desirable. Methods currently utilized for the identification of sites of DVT are contrast venography and compression B-mode ultrasound; the choice of which technique is used depends on the expected location of the thrombus. The currently-available techniques are unsatisfactory, however, because in many cases they yield inaccurate results, are uncomfortable for the patient or are otherwise clinically inappropriate. The technique of contrast venography is additionally disadvantageous because it is an invasive technique.
Current methods used to diagnose PE include chest X-ray, electrocardiogram (EKG), arterial oxygen tension, perfusion and ventilation lung scans, and pulmonary angiography. However, none of these methods (except pulmonary angiography) is capable of providing an unequivocal diagnosis, and this method is an invasive method.
One area of medicine concerned with the non-invasive localization of pathologies in vivo is nuclear medicine. In the field of nuclear medicine, pathological conditions are localized, or their extent is assessed, by detecting the specific distribution of small quantities of internally-administered radioactively labeled tracer compounds (called radiotracers, radiopharmaceuticals or scintigraphic imaging agents). Methods for detecting these radiopharmaceuticals are known generally as scintigraphic imaging, radioimaging or radiodiagnostic imaging methods. A variety of radionuclides are known to be useful for radioimaging, including .sup.18 F, .sup.64 Cu, .sup.67 Ga, .sup.68 Ga, .sup.99m Tc, .sup.111 In, .sup.123 I, .sup.125 I and .sup.131 I. Of these radionuclides, Tc-99m and In-111 are preferred single photon-emitting radionuclides and Ga-68 is a preferred positron-emitting radionuclide.
A number of factors must be considered for optimal radioimaging in humans. To maximize the efficiency of detection, a radionuclide that emits gamma energy in the 100 to 200 keV range is preferred. To minimize the absorbed radiation dose to the patient, the physical half-life of the radionuclide should be as short as the imaging procedure will allow. To allow for examinations to be performed on any day and at any time of the day, it is advantageous to have a source of the radionuclide always available at the clinical site. Tc-99m is a preferred radionuclide for radioimaging in humans because it emits gamma radiation at 140 keV, it has a physical half-life of 6 hours, and it is readily available on-site using a molybdenum-99/technetium-99m generator.
A gamma-emitting radiotracer that binds specifically to a component of a thrombus in preference to other tissues when administered in vivo would provide an external scintigraphic image to define the location of the thrombus-bound radiotracer and hence the thrombus. Such a radiotracer should strongly and specifically bind to a component of a thrombus in order to achieve specific radioimaging of thrombus sites. The primary components of thrombi to which such a radiotracer might specifically bind are blood cells, largely activated platelets, enmeshed in cross-linked fibrin.
Attempts to provide radiotracers for imaging thrombi are known in the prior art. These include autologous platelets, labeled with either In-111 or Tc-99m, and I-123 and I-125 labeled fibrinogen (the latter detected with a gamma scintillation probe as opposed to a gamma camera). Additional radiolabeled compounds used to label thrombi include plasmin, plasminogen activators, heparin, fibronectin, fibrin Fragment E.sup.1 and anti-fibrin and anti-platelet monoclonal antibodies (see Knight, 1990, Sem. Nucl. Med. 20: 52-67 for review).
Activated platelets are particularly good targets for radioimaging thrombi because they are not normally found in circulating blood (which contains unactivated platelets). Activated platelets express the GPIIb/IIIa receptor on their cell surfaces which binds to fibrinogen (Parise & Phillips, 1985, J. Biol. Chem. 260: 10698-10707). However, small, synthetic analogues have been developed that bind to this receptor (see, for example, Klein et al., 1992, U.S. Pat. No. 5,086,069 and Egbertson et al., 1992, European Patent Application No. EPA 0478328A1). Some of these synthetic molecules bind to the GPIIa/IIIb receptor with very high affinity (see Egbertson et al., ibid.).
Compounds having the ability to bind to the platelet GPIIb/IIIa receptor are known in the prior art.
Ruoslahti & Pierschbacher, U.S. Pat. No. 4,578,079 describe peptides capable of binding to platelets.
Ruoslahti & Pierschbacher, U.S. Pat. No. 4,792,525 describe peptides capable of binding to platelets.
Klein et al., 1992, U.S. Pat. No. 5,086,069 disclose guanidine derivatives that bind to the GPIIb/IIIa receptor.
Pierschbacher et al., 1989, International Patent Application Ser. No. PCT/US88/04403 disclose conformationally-restricted RGD-containing peptides for inhibiting cell attachment to a substratum.
Alig et al , 1989, European Patent Application 89122396.8, disclose benzoic acid and phenylalanine derivatives as fibrinogen antagonists.
Nutt et al., 1990, European Patent Application 90202015.5 disclose cyclic RGD peptides that are fibrinogen receptor antagonists.
Nutt et al., 1990, European Patent Application 90202030.4 disclose cyclic RGD peptides that are fibrinogen receptor antagonists.
Nutt et al., 1990, European Patent Application 90202031.2 disclose cyclic RGD peptides that are fibrinogen receptor antagonists.
Nutt et al., 1990, European Patent Application 90202032.0 disclose cyclic RGD peptides that are fibrinogen receptor antagonists.
Nutt et al., 1990, European Patent Application 90311148.2 disclose cyclic peptides that are fibrinogen receptor antagonists.
Nutt et al., 1990, European Patent Application 90311151.6 disclose cyclic peptides that are fibrinogen receptor antagonists.
Ali et al., 1990, European Patent Application 90311537.6 disclose cyclic peptides that are fibrinogen receptor antagonists.
Barker et al., 1991, International Patent Application Ser. No. PCT/US90/03788 disclose cyclic peptides for inhibiting platelet aggregation.
Pierschbacher et al., 1991, International Patent Application Ser. No. PCT/US91/02356 disclose cyclic peptides that are fibrinogen receptor antagonists.
Egbertson et al., 1992, European Patent Application 0478328A1 disclose tyrosine derivatives that bind with high affinity to the GPIIb/IIIa receptor.
Duggan et al, 1992, European Patent Application 92304111.5 disclose fibrinogen receptor antagonists.
Garland et al, 1992 European Patent Applications 92103861.8 and 92108214.5 disclose phenylamide derivatives as platelet aggregation inhibitors.
Bondinell et al, 1993, International Patent Application Ser. No. PCT/US92/05463 disclose bicyclic fibrinogen antagonists.
Blackburn et al., 1993, International Patent Application Ser. No. PCT/US92/08788, disclose nonpeptidyl integrin inhibitors having specificity for the GPIIb/IIIa receptor.
Hartman et al., 1993, European Patent Application Ser. No. 93309924.6 disclose fibrinogen receptor antagonists.
Ojima et al., 1992, 204th Meeting, Amer. Chem. Soc. Abst. 44 disclose synthetic multimeric RDGF peptides useful in inhibiting platelet aggregation.
Hartman et al., 1992, J. Med. Chem. 35: 4640-4642 describe tyrosine derivatives that have a high affinity for the GPIIb/IIIa receptor.
Radiolabeled peptides for radioimaging thrombi also have been reported in the prior art.
Stuttle, 1990, International Patent Application Ser. No. PCT/GB90/00933 discloses radioactively labeled peptides containing from 3 to 10 amino acids comprising the sequence arginine-glycine-aspartic acid (RGD), capable of binding to an RGD binding site in vivo.
Rodwell et al., 1991, International Patent Application Ser. No. PCT/US91/03116 disclose conjugates of "molecular recognition units" with "effector domains".
Maraganore et al., 1991, International Patent Application Ser. No. PCT/US90/04642 disclose a radiolabeled thrombin inhibitor comprising (a) a inhibitor moiety; (b) a linker moiety; and (c) an "anion binding exosite (ABE)" moiety.
The use of chelating agents for radiolabeling peptides, and methods for labeling peptides with Tc-99m are known in the prior art and are disclosed in co-pending U.S. patent applications Ser. Nos. 07/653,012, now abandoned, which issued as U.S. Pat. No. 5,654,272; 07/807,062, now U.S. Pat. No. 5,443,815; 07/871,282, a divisional of which issued as U.S. Pat. No. 5,720,934; 07/886,752, now abandoned, a continuation of which has been allowed as U.S. Ser. No. 08/273,274; 07/893,981, now U.S. Pat. No. 5,508,020; and radiolabeled peptides for use as scintigraphic imaging agents for imaging thrombi are known in the prior art and are disclosed in co-pending U.S. patent applications Ser. Nos. 07/886,752, now abandoned, a continuation of which has been allowed as U.S. Ser. No. 08/273,274; 07/893,981, now U.S. Pat. No. 5,508,020; and 08/044,825, now abandoned, which issued as U.S. Pat. No. 5,645,815 which are hereby incorporated by reference.
There remains a need for small (to enhance blood and background tissue clearance), synthetic (to make routine manufacture practicable and to ease regulatory acceptance), high-affinity, specific-binding molecules radiolabeled with a convenient radiolabel, preferably Tc-99m, for use in imaging thrombi in vivo. Small synthetic compounds that bind specifically to the GPIIb/IIIa receptor expressed on the cell surface of activated platelets, and that are radiolabeled with a convenient radioisotope, preferably Tc-99m, fulfill this need in the art.